PROJECT SUMMARY There is a fundamental gap in understanding how hematopoietic stem cells (HSCs) first emerge during embryonic development. This includes 1) the identity of the HSC precursor (pre-HSCs), and 2) why HSCs (but not pre-HSCs) are able to seed the bone marrow and engraft upon transplantation into adult hosts. Without first addressing these questions, the potential to apply that knowledge to improve the engraftment of HSCs, or pluripotent stem cell (PSC) derived HSCs, upon transplantation as treatments for blood diseases will remain unrealized. The long-term goal of this research team is to better understand the molecular mechanisms that regulate HSC emergence and maturation in the embryo. The overall objective in this application is to identify pre-HSCs in the embryo and determine why they cannot engraft in adults. The central hypothesis is that an embryonic population called ?eKLS? contains pre-HSCs that cannot engraft into bone marrow due to the absence of homing molecules on their surface, such as the chemokine receptor Cxcr4. This hypothesis has been formulated on the basis of two lines of preliminary data: First, that sorted eKLS cells can engraft and become HSCs when transplanted into neonatal mice, and second, that eKLS cells lack expression of Cxcr4 and other homing factors. The rationale for the proposed research is that, once pre-HSCs have been identified, they can be compared to adult HSCs to better understand what factors are critical for engraftment. This hypothesis will be tested by pursuing two specific aims: 1) To determine whether eKLS cells or another embryonic cell gives rise to HSCs in vivo, and 2) To determine whether the ability to respond to Cxcr4/Cxcl12 signaling distinguishes pre-HSCs from HSCs. Under the first aim, the neonatal transplantation system will be used to identify which embryonic populations and in which embryonic tissues contain pre-HSCs. Under the second aim, Cxcr4 and other homing factors will be expressed in eKLS cells to determine whether they can imbue those cells with the ability to engraft in adult recipients. The research proposed in this application is conceptually innovative because it focuses on the differences between HSCs and their immediate precursors as a way to understand how HSCs emerge and mature during embryonic development. The proposed research is significant because it could lead to breakthroughs in the engraftabilty of HSCs and/or PSC-derived HSCs upon transplantation. This could dramatically improve the safety and efficacy of HSC transplantation as a treatment for nearly any blood disease.